Abstract P. aeruginosa strains differ from one to another in their ability to cause severe disease, in both animals and plants. Since about 90% of P. aeruginosa ORFs, including a large number that encode virulence factors, make up a core set of genes that are conserved throughout all strains, it is likely that a proportion of the phenotypic heterogeneity of P. aeruginosa is attributable to large strain-specific genomic islands that were acquired through horizontal transfer. These genomic islands represent a wealth of genetic information that remains relatively unexplored. Although some genomic islands have been associated with virulence in various models (and therefore are classified as pathogenicity islands), the full impact of variable genomic islands on the virulence of P. aeruginosa has yet to be examined. We hypothesize that genomic islands present in some strains but absent in others account for some of the heterogeneity in virulence of P. aeruginosa strains and that characterization of these genomic islands will result in the identification of novel virulence determinants. The goals of this proposal are two-fold: first, a substantial number of novel genomic islands will be identified and characterized, adding to the database of genetic information known to be carried by this bacterium and furthering our understanding of the evolution of these genetic elements. Second, genomic islands that contribute to the overall virulence of P. aeruginosa will be identified, as will their individual ORFs that account for this phenotype. The general strategy of this proposal is to exploit the differences in virulence between P. aeruginosa strains to identify strains with a high likelihood of containing large amounts of variable genetic information in the form of novel genomic islands. These strains will be targeted for in-depth analysis. Genomic islands in these strains will be sequenced, and those that contribute to virulence will be identified. Finally, mutational analyses will be performed to identify the ORFs harbored by these islands that are responsible for the enhanced virulence. Successful completion of this proposal will yield a wealth of information regarding the content and characteristics of P. aeruginosa genomic islands and will identify novel virulence factors. Clinically, the identification of genomic islands that impact the disease-causing potential of P. aeruginosa strains is of potential prognostic and therapeutic importance.